Why low fold change (treatment vs. control)? - (Dec/07/2008 )

Hi everybody,

My western results are generally ok (mostly clear bands, consistent) but the biggest fold change is only about 1.7 (treatment vs. control)! I'm disappointed and I'm sure my boss will not be happy with it!Beside signal saturation issue during film development, what steps could lower the fold change I want to see (if assume a bigger change in the real situation)? Composition of lysis buffer, dilution of antibodies? For example, many people seem to agree that 4C overnight incubation with Ab is better than room temperature for 1 hour? Better in what sense (stronger signal or better distinguishing power for changes)? For my case, signal is already strong and I want to avoid the under-estimation of the changes I expect. Another Q: I'm doing 7 time points on the same gel (control+treatment), so plus the ladder, I'm making full use of the 15-# gel, but it seems that the last band was bigger and awry. Is it really hard to get beautifyl results when making full use of the 15 wells?

Thanks in advance!

Hanhan

-hanhan2008-

Hey,

Did you try loading less sample on the gel? If you load less you will reduce the saturation of the signal.Hope this helps!

-NatB-

Thank you! I loaded 10 ug usually, 5 ug last time. Signal was still pretty strong. HO-1, Actin and secondary Ab's were all 1:10000. When using 3 sec exposure, I did get some bands that seemingly with no saturation issue, but still the fold change is low. That's why I wonder if there are other factors that I neglected.

QUOTE (NatB @ Dec 8 2008, 12:45 AM)

Hey,

Did you try loading less sample on the gel? If you load less you will reduce the saturation of the signal.Hope this helps!

-hanhan2008-

Western blots are more qualitative than quantitative -- I'd be skeptical of a paper that tried to assign fold-change numbers on the basis of a western blot....

-HomeBrew-

Yeah, I agree. In reality, a lot of papers show fold changes and my task is even more challenging: a fold change change over time at a certain dose, dose-response curves when having multiple doses, and finally comparison between these dose-response curves.

QUOTE (HomeBrew @ Dec 8 2008, 09:20 PM)

Western blots are more qualitative than quantitative -- I'd be skeptical of a paper that tried to assign fold-change numbers on the basis of a western blot....

-hanhan2008-

QUOTE (HomeBrew @ Dec 8 2008, 06:20 PM)

Western blots are more qualitative than quantitative -- I'd be skeptical of a paper that tried to assign fold-change numbers on the basis of a western blot....

True...WB only allows you to have a glimpse on change, in case of same loading amount.